Protein-mediated membrane fusion is essential for maintaining eukaryotic cell organization and propagation of major human viruses. Many clinically relevant members of the paramyxovirus family rely on the concerted action of two envelope glycoproteins, the attachment and fusion protein, to fuse their envelope with the target cell plasma membrane for cell entry. However, despite their clinical importance, fundamental mechanistic principles that govern the organization and function of native paramyxovirus fusion complexes are not understood. Towards the overarching goal of elucidating these principles, this project will pursue the problem in three basic questions: What is the spatial organization of the envelope glycoprotein hetero-oligomer complexes in the native, metastable conformation displayed on the surface of infectious particles? How does receptor binding affect attachment protein organization? What is the molecular mechanism that links receptor binding with fusion protein refolding into the thermodynamically stable postfusion conformation? With crystal structures of isolated ectodomains of different paramyxovirus glycoproteins at hand, the proposed studies will focus on measles virus envelope glycoproteins to address these questions in a comprehensive, interdisciplinary approach that interfaces innovative imaging, biochemical, functional, and computational experimental strategies. Cryo-electron tomography combined with envelope glycoprotein engineering will elucidate the overall spatial organization of hydrated fusion complexes displayed on the surface of viral particles, alone and after treatment with soluble receptor (aim 1). Native gel electrophoresis, H oligomer stabilizing and destabilizing modifications, and H bimolecular complementation will extract functional information by assessing the effect of receptor binding on attachment protein organization and characterizing the molecular nature of the signal that initiates fusion protein refolding (aim 2). Molecular modeling-guided mutagenesis, biochemical contact domain mapping, peptide binding and mass spectrometry will cross-examine, expand and functionally characterize candidate intermolecular contacts found in pilot studies, resulting in the identification of discrete microdomains in either glycoprotein that line the hetero-oligomer interface and control the structural integrity of a native paramyxovirus fusion complex (aim 3).